The isolated PBMCs were washed with pre-cold PBS and cultured with supplemented RPMI 1640 medium in 24-well plates. Cells were stimulated with rhBAFF (20 ng/ml, R&D Systems, Minneapolis, MN, USA) or a combination of rhBAFF and BR3-Fc (100μg/ml, R&D Systems, Minneapolis, MN, USA) at 37 °C with 5% CO2 for 48 h. Cells were harvested for assessment of apoptosis of CD4+ and CD8+ cells using flow cytometry. Isolated CD4+ or CD8+ cells were cultured with supplemented RPMI 1640 medium and phytohemagglutinin (Sigma-Aldrich and Merck KGaA, Darmstadt, Germany) in 24-well plates. Cells were stimulated with rhBAFF or a combination of rhBAFF and BR3-Fc at 37 °C with 5% CO2 for 72 h. For CD4+ cells culture, the supernatant and cells were seperately obtained for the detection of IL-4 and IFN-γ expression. For CD8+ cells culture, the cells were harvested for the detection of mRNA expression of perforin and granzyme B.
Rhbaff
Manufactured by R&D Systems
Sourced in United States
RhBAFF is a recombinant human B-cell activating factor (BAFF) protein. BAFF is a member of the tumor necrosis factor (TNF) ligand family and plays a critical role in B-cell survival and maturation.
Automatically generated - may contain errors
Lab products found in correlation
Cpg odn 2006, by InvivoGen (2 mentions)
Rhtgf β, by R&D Systems (2 mentions)
Rhapril, by R&D Systems (2 mentions)
Rhcd40l, by R&D Systems (2 mentions)
Cd19 beads, by Miltenyi Biotec (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Br3 fc, by R&D Systems (1 mentions)
Anti cd40, by BD (1 mentions)
Negative selection, by Miltenyi Biotec (1 mentions)
Human il 10, by Thermo Fisher Scientific (1 mentions)
Human il 2, by Thermo Fisher Scientific (1 mentions)
Ultra low igg fbs, by Thermo Fisher Scientific (1 mentions)
Cd27 microbeads, by Miltenyi Biotec (1 mentions)
Pierce jacalin agarose, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Human b cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Facscalibur, by BD (1 mentions)
Rosettesep, by STEMCELL (1 mentions)
5 protocols using rhbaff
1
Evaluating BAFF-Induced T Cell Responses
2
Naïve B Cell Differentiation
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Cells are cultured in IMDM supplemented with 10% FCS, 100 IU/ml penicillin and 50 μg/ml streptomycin. PBMCs derived from healthy donors were isolated using ficoll-Paque. CD19+ B cells were subsequently MACS-isolated using CD19 beads (Miltenyi Biotec), where after CD19+IgD+CD27− naïve B cells were FACS-sorted. 2 ×105 Naïve B cells were cultured for 6 days on 50.000 irradiated CD40L-expressing fibroblasts in the presence of 50 ng/ml recombinant mouse IL-21, 0,5 ng/ml rhTGF-β (R&D Systems), 100 ng/ml rhAPRIL (R&D Systems) and 100 ng/ml rhBAFF (R&D Systems). When stated, blocking antibodies to TACI and BCMA (both 10 μg/ml, R&D systems) were added to the cultures. In some experiments, B cells were additionally stimulated for 2 days with 10 ng/ml LPS (Invivogen), 10 μg/ml CpG (ODN2006, Invivogen) or medium as control after the 6 day culture protocol.
3
Western Blot Analysis of NF-κB Pathway
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p52/p100 and β-actin were monitored by western blot as described previously31 (link). Briefly, p52 processing was detected after 6 or 18 h of stimulation. Mouse and human RPTEC cells were stimulated with recombinant mouse or human TWEAK (100 ng/mL, R&D Systems). Primary B cells were isolated by negative selection (Miltenyi) from either mouse spleen or human whole blood. Human primary B cells were stimulated with either rhCD40L (10 μg/mL) or rhBAFF (400 ng/mL) (R&D Systems). Mouse primary B cells were stimulated with either anti-CD40 (10 μg/mL, BD Biosciences) or rmBAFF (400 ng/mL). After 6 or 18 h, whole cell extracts were generated using RIPA buffer (with protease and phosphatase inhibition), and approximately 10 μg of total protein was monitored for p52/p100 and β-Actin by western blot. HeLa cells (ATCC) were stimulated for 8 h with anti-LTβR agonist antibody and cytoplasmic and nuclear extracts were generated using kits purchase from Thermo Scientific and approximately 10 mg of each extract was monitored for p52/p100, phospho-RelA, RelA, or β-Actin, Histone H3, or Lamin B1 controls. Uncropped western blots, as required by journal policies, are provided in Supplementary Fig. 13 and 14 .
4
Hybridoma Generation for Anti-PV IgA Antibodies
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Hybridoma methods were performed with cultured PBMCs as described previously, with minor modifications [32 (link)]. PBMCs were isolated using ficoll-Paque (17-1440-02; GE Healthcare, Chicago, IL, USA) density gradient centrifugation and cryopreserved. CD27+ cells were MACS-isolated using CD27 microbeads (130-051-601; Miltenyi Biotec, Cambridge, MA, USA). CD27+ cells (2 × 105 cells/well) were plated in a 24-well plate and cultured for eight days in advanced RPMI supplemented with 10% FBS, 100 IU/mL penicillin, 50 μg/mL streptomycin, 5 ng/mL human IL-2, 50 ng/mL human IL-10 (Peprotech, Rocky Hill, NJ, USA), 10% CHO cell conditioned medium containing Ultra CD40L (Multimeric Biotherapeutics, La Jolla, CA, USA), 1 μg/mL CpG (ODN2006, Invivogen, San Diego, CA, USA), 100 ng/mL rhBAFF, 1 μg/mL cyclosporin, 0.5 ng/mL rhTGF-β and 100 ng/mL rhAPRIL (R&D Systems, Minneapolis, MN, USA). On day 8, cells were harvested and fused with the LCX OCMS fusion partner cell line by electrofusion [33 (link)]. Positive wells were subjected to three rounds of single cell cloning to establish stable hybridomas secreting anti-PV IgA mAbs. For scale up, hybridoma clones were adapted to 5% Ultra Low IgG FBS (Thermo Fisher Scientific, Waltham, MA, USA) and purified with columns packed with Pierce Jacalin Agarose (20395; Thermo Fisher) following the manufacturer’s recommendations.
5
Modulating ICOSL Expression in Activated B Cells
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Human B cells were isolated from whole blood. B cells were enriched by RosetteSep protocol (Stem Cell Technologies), and then isolated using Human B cell isolation kit II (Miltenyi). A total of 0.2 × 106 B cells were cultured and stimulated with rhCD40L (5 μg/mL, 48 h, R&D Systems), or rhBAFF (100 ng/mL, 72 h, R&D Systems) in the presence or absence of a NIK SMI1 or NIK SMI2 dilution series (10 μM – 1.5 nM). Mean fluorescent intensity (MFI) of CD40L and BAFF induced ICOSL expression was measured by flow cytometry on cells stained with anti-CD20 and anti-ICOSL, using a FACS Calibur instrument (BD). Plots and IC50 measurements were generated using GraphPad Prism Software.
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